Axons and dendrites contain elaborate cytoskeletons that consist of microtubules, neurofilaments, and microfilaments. These cytoskeletal structures comprise an architectural framework that defines the external shape of the axon and the dendrite. Thus, the mechanisms that organize the cytoskeleton in neurons contribute directly to the elaboration and maintenance of neuronal form and thereby function. This application proposes direct experiments on the dynamic processes that organize the cytoskeleton in growing axons and dendrites. These experiments focus primarily on microtubules (MTs). MTs are linear polymers of tubulin subunits. Tubulin is synthesized in the neuron cell body and is then conveyed into the axon and possibly the dendrite by active transport processes. The major goal of the proposed experiments is to understand how tubulin transport mechanisms and local assembly mechanisms cooperate to shape the MT array during axonal and dendritic growth. Toward this end, experiments are proposed to (1) quantify parameters of MT dynamics in growing neurites, (2) identify the stable templates that nucleate MT assembly in axons and dendrites, and (3) dissect the mechanism that transport MT proteins in growing neurites. All of these experiments combine microinjection of haptinized tubulin into cultured neurons with quantitative digital image processing and analysis methods. We have already developed much of the required methodology and have begun to use these approaches to quantify aspects of MT dynamics in growing axons. Successful completion of the proposed experiments will define essential mechanisms involved in generating the MT arrangements required for the normal growth of axons and dendrites. In addition, many pathologies of the nervous system are characterized by abnormalities of cytoskeletal organization. By defining normal mechanisms for generating and maintaining MT arrangements in neurons, the proposed research will contribute toward a better understanding of these pathologies.